Boring systematics: A genome skimmed phylogeny of ctenostome bryozoans and their endolithic family Penetrantiidae with the description of one new species

Abstract Ctenostomes are a group of gymnolaemate bryozoans with an uncalcified chitinous body wall having few external, skeletal characters. Hence, species identification is challenging and their systematics remain poorly understood, even more so when they exhibit an endolithic (boring) lifestyle. Currently, there are four Recent families of endolithic bryozoans that live inside mineralized substrates like mollusk shells. In particular, Penetrantiidae Silén, 1946 has received considerable attention and its systematic affinity to either cheilostomes or ctenostomes has been debated. Species delimitation of penetrantiids remains difficult, owing to a high degree of colonial and zooidal plasticity. Consequently, an additional molecular approach is essential to unravel the systematics of penetrantiids, their phylogenetic placement and their species diversity. We therefore sequenced the mitochondrial (mt) genomes and two nuclear markers of 27 ctenostome species including nine penetrantiids. Our phylogeny supports the Penetrantiidae as a monophyletic group placed as sister taxon to the remaining ctenostomes alongside paludicellids, arachnidioids and terebriporids. The boring family Terebriporidae d'Orbigny, 1847 were previously considered to be among vesicularioids, but our results suggest an arachnidioid affinity instead. Ctenostome paraphyly is supported by our data, as the cheilostomes nest within them. A Multiporata clade is also well supported, including the former victorelloid genus Sundanella. Altogether, this study provides new insights into ctenostome systematics, assists with species delimitation and contributes to our understanding of the bryozoan tree of life.


| INTRODUC TI ON
Bryozoa is a clade of sessile and filter feeding metazoans that occurs in marine and freshwater habitats.As epibenthic organisms, they colonize various hard substrates and can create colonies up to several centimeters in size, which are composed of individual units (zooids) (Ryland, 1970;Schwaha, 2020a).Zooids that contain a tentacle crown (lophophore and digestive system) and actively feed are called autozooids, while polymorphic zooids specialized for reproduction, defense or colonial connectivity are called heterozooids (Mukai et al., 1997;Schack et al., 2019).The majority of the currently known extant ~6000 bryozoan species (Bock & Gordon, 2013) thrive in marine environments from intertidal to subtidal regimes and to depths of more than 7000 m (Grischenko et al., 2019;Ryland, 1970).
Because only a few external characters can be detected, correct species identification of penetrantiids remains challenging and some species were described based on their boring traces alone.
Although the number of molecular phylogenetic studies on bryozoans has increased in recent years, most of them focused on cheilostome bryozoans, with only a few studies included ctenostome representatives (Fuchs et al., 2009;Orr et al., 2019Orr et al., , 2021Orr et al., , 2022;;Waeschenbach et al., 2012Waeschenbach et al., , 2015)).The most comprehensive ctenostome molecular phylogenies are based on a handful of genes but play a crucial role in our understanding of ctenostome systematics and support their paraphyly (Waeschenbach et al., 2012(Waeschenbach et al., , 2015)).
Consequently, this study seeks for a larger phylogenetic analysis based on data from mitochondrial (mt) genomes and nuclear ribosomal RNA (rRNA) genes 18S and 28S.Our analysis includes 27 ctenostome species representing seven of their eight superfamilies.
The molecular phylogenetic framework is combined with known morphological characters to shed light on (1) general ctenostome phylogeny, (2) the systematic position of the Penetrantiidae and (3) the interrelationships of Penetrantiidae.Additionally, this study aims to unravel potential cryptic species complexes in the genus Penetrantia by including specimens from ten different geographical regions.Furthermore, we include one species of the boring ctenostome family Terebriporidae to evaluate whether an endolithic lifestyle evolved independently within ctenostomes.As this is one of the first studies comprising such a large molecular dataset of ctenostome bryozoans, it will also contribute to future analysis on the systematics of Gymnolaemata and might help to resolve the origin of cheilostomes and the paraphyly of ctenostomes.

| Sample collection and imaging
Twenty-seven specimens from 11 different localities were collected for genome skimming including nine different Penetrantia and 18 additional ctenostome specimens (Table 1).One additional penetrantiid specimen (Penetrantia sp.) was collected in Helgoland, Germany (54°08.339′N 7°52.298′E) for sanger sequencing of the cytochrome c oxidase subunit I (cox1) gene (OR632352) and genetic distance analysis only (see below).Samples were either collected in the intertidal zone by hand or in shallow subtidal areas by TA B L E 1 Sample details and accession numbers of specimens used for genome skimming in this study.

| DNA extraction
Genomic DNA (gDNA) of all samples was extracted using the QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany) following the manufacturer's guidelines.Specimens of the endolithic genera Penetrantia and Terebripora were removed from their calcareous substrate either by mechanical breakage or by dissolving the substrate with 20% ethylenediaminetetraacetic acid (EDTA).

| PCR amplification, sequencing and cox1 gene sequence analysis
Prior to genome skimming, the cox1 gene was sequenced for each specimen using PCR and Sanger sequencing.PCR amplification used universal (Folmer et al., 1994) or specific bryozoan primers (Table A1).

| Illumina sequencing, assembly and annotation
Library preparation and sequencing were conducted by the Next- and 127.Mt genome contigs were identified using BLASTN (Altschul et al., 1990) and annotated with the MITOS2 web server (Donath et al., 2019) using the metazoan reference database RefSeq 63 and the invertebrate genetic code.Circularized mitochondrial genome maps (Figure A1) were generated with OrganellarGenome-DRAW (OGDRAW) online server v 1.3.1 (Greiner et al., 2019).Manual curation of the mitogenomes was undertaken using previously published mitogenomes of bryozoans available on NCBI as references.
In cases where incomplete mitogenome contigs were not recovered, Exonerate v2.4.0 (Slater & Birney, 2005) with the affine: local model and maximum intron length set to 40 kb was used to scan the remaining contigs in the assemblies to identify any missing mt genes (13 protein-coding genes [PCG] and 12S and 16S rRNA genes; transfer RNAs were not scanned).18S and 28S rRNA genes were annotated using RNAmmer (Lagesen et al., 2007).
Phylogenetic trees were constructed using Bayesian inference (BI) and maximum likelihood (ML) on a mixed partitioned data matrix including 16 partitions (12 PCGs, two mt rRNA genes (12S and 16S) and two nuclear rRNA genes (18S and 28S)).Mt PCGs were processed as amino acids while mt rRNA and nuclear rRNA genes as nu-  et al., 2017).The convergence was also assed based on the average standard deviation of split frequencies (ASDOSF) and was <0.01 (0.000034)"The first 25% of samples were discarded as burn-in, and the remaining trees were used to calculate posterior probability values and to build the consensus tree.The final ML and BI trees were visualized and adjusted in Figtree v1.4.4 (http:// tree.bio.ed.ac.uk/ softw are/ figtr ee/ ).

| Genetic distance
ML-corrected substitutions per site were calculated in MEGA 7 using the maximum composite likelihood parameter with a gamma parameter of 1.0 (Kumar et al., 2008;Tamura et al., 2004Tamura et al., , 2021)).

| Alignment
We generated sequences of 27 specimens that belong to 25 morphospecies and successfully assembled and annotated all PCGs, two rRNAs and two nuclear rRNA genes of 20 specimens while the atp8 gene was not recovered in seven samples (Table A2).As a result, the atp8 gene was excluded from our final data matrix, together with 18S and 28S of Terebripora sp.The remaining sequences of these 27 samples were combined with published sequences of the ctenostome Monobryozoon ambulans (Schwaha et al., 2024), nine cheilostome species (Orr et al., 2021) and the phylactolaemate Pectinatella magnifica as outgroup (Fuchs et al., 2009;Gim et al., 2018;Waeschenbach et al., 2009; Table A2).
Our data matrix included 16 genes (12 mt PCGs, two mt rRNA and two nuclear rRNA genes) totaling 9702 characters (2834 amino acids and 6868 nucleotide sites).

| Ctenostome phylogeny and placement of Penetrantia
The ML tree, which is based on the complete data matrix, is shown in Figure 1.Highly consistent tree topologies were observed from both phylogeny reconstruction methods (ML, Figure 1  .Both taxa form a highly supported sister-group relationship (80 BS/0.99 PP).Within Victorellidae, Tanganella muelleri is the sister taxon to Bulbella abscondita and Amphibiobeania epiphylla.Among Vesicularioidea, Amathia gracilis is the sister taxon to the paraphyletic assemblage of Amathia with the inclusion of Vesicularia.With moderate support (72 BS/1.00 PP), Amathia ernsti and Vesicularia spinosa cluster together with Amathia distans as sister taxon (Figure 1, "Ctenosotmata" C).  the cox1 genetic divergence is 21.9%, confirming them to be different species.Contrary, P. concharum specimens from Sweden and

| Interrelationships of Penetrantia and their genetic distances
France exhibit a genetic distance of 0.3% (Figure 2; Table A3).
Penetrantia sp. from France (Roscoff) is also confirmed in Germany (Helgoland) with a genetic divergence of 2.3% based on the barcoding region of the cox1 gene (Table A4).
The parva complex forms a monophyletic clade with high support (90 BS/1.00 PP) and all three representatives exhibit the species-specific aperture outline with prominent apertural notches (Figure 2).Penetrantia parva from the northern Island of New Zealand is the sister taxon to a clade represented by P. cf. parva from the southern Island of New Zealand and P. cf. parva from Chile with moderate support (91 BS/1.00 PP; Figure 2).The cox1 genetic distances between representatives of this complex are: northern and southern Islands of New Zealand -12.1%;P. parva from northern New Zealand and P. cf. parva from Chile -12.9%; southern New Zealand and Chile -9.8% (Table A3).  in width, dome-shaped in cross section, rough crescent area on frontal-oral side, partially composed of calcium carbonate (Figure 3f, g).Multiple brown bodies common (Figure 3d).Gonozooid same length as autozooid, brood chamber on anal side about 230 μm long, pear-shaped in longitudinal section.Gonozooidal tube longer than brood chamber with slandered basal tip, bending in anal direction.
Etymology.Japonica is the adjective in the feminine case, referring to Japan, which represents the type locality of this possibly endemic species.
Remarks.The operculum morphology is very similar between P. japonica sp.nov., P. clionoides Smyth, 1988 and Penetrantia bellardiellae Schwaha, 2019.The opercula of these three species are composed of calcium carbonate, with a rough crescent-shaped area on the frontal side and thereby tells them apart from all other penetrantiids.In contrast to P. clionoides and P. bellardiellae, P. japonica sp.nov.has the largest tubulet intervals and unique gonozooids with a lean basal tip which bends slightly in frontal direction.
Paludicella articulata lacks such cystid appendages, has a unique cruciform branching pattern and is restricted to freshwater habitats.These major differences led to the placement of Paludicella into the separate superfamily Paludicelloidea (Jebram, 1973;Schwaha, 2020c;Todd, 2000).The family Terebriporidae is one of four Recent endolithic ctenostome families and was considered a vesicularioid ctenostome with true stolonate colonies, and the presence of a gizzard was previously reported (Schwaha, 2020c;Soule & Soule, 1969).However, this information about the soft body morphology in terebriporids remains doubtful (Pohowsky, 1978).
Subsequent histological analyses are necessary to clarify whether terebriporids have kenozooidal stolons or arachnidioid-like cystid appendages and if they feature a gizzard or not (Pohowsky, 1978).
Since this clade shows only a moderate support and a different topology within the BI analysis, a denser taxon sampling that includes more members of the Arachnidioidea, e.g., Immergentiidae and Nolellidae, is required to better resolve the unexpected sistergroup relationship of Paludicella with Arachnidium and Terebripora.
The fourth family within clade A, Penetrantiidae, possess many distinct characters (e.g., operculum and kenozooidal stolons) that are not present in Paludicellidae or Arachnidioidea and will discussed in more detail later (see below) (Jebram, 1973;Schwaha, 2020c).
The second main clade (B) represents the superfamily Alcyonidioidea, a taxon characterized by tightly arranged zooids that are always in close contact with the body wall of neighboring zooids and never by stolon-like connections (Schwaha, 2020c).Unlike other studies (Jebram, 1986;Todd, 2000;Waeschenbach et al., 2012), our phylogenetic analysis does not support alcyonidioids as sister taxon to all remaining ctenostomes but of clade A instead (Arachnidiids, Paludicellids and Penetrantiids).Consequently, our study indicates that the serial arrangement of zooids as found in paludicellids, arachnidioids and penetrantiids could represent the ancestral colony structure of ctenostomes rather than simple encrusting sheet-like colonies of alcyonidioideans as previously suggested (Jebram, 1973;Schwaha, 2020c).Certainly, a larger taxon sampling within both clades might alter the phylogeny, since arachnidioids are only represented by one species in this study.
The only superfamily not included in the current study is Benedeniporoidea and was previously considered the sister taxon to all remaining ctenostomes.This led to the establishment of the "Protoctenostomata" -"Euctenostomata" concept, with

Benedeniporoidea as early protoctenostome and all remaining
Recent ctenostomes belonging to euctenostomes (Jebram, 1973;Todd, 2000).However, this phylogenetic hypothesis would imply that a ctenostome-like ancestor possessed serially erect colonies, which is a rare state among Recent ctenostomes.Additionally, species of this superfamily were only rarely found and detailed information on their morphology as well as sequence data is missing (Schwaha, 2020c).
Multiporata, a recently erected taxon of alcyonidioid bryozoans that is characterized by multiporous pore plates, is monophyletic and nests within Alcyonidioidea.These distinct pore-plates are usually known from cheilostomes and not found in other ctenostome bryozoans (Schwaha, Winston, et al., 2022).The multiporate genera | 11 of 23 Flustrellidra and Pherusella are sister taxa in our analysis and share some specific characters, e.g., a rectangular to bilateral shaped orifice and pseudocyphonautes larvae.The latter is only present in these two families and resembles an apomorphy of this group (Decker et al., 2020(Decker et al., , 2021;;Reed, 1991).This close relationship was also shown by a recent phylogenomic study (Saadi et al., 2022).Our study supports the affiliation of sundanellids to Multiporata and not to Victorelloidea.This affiliation is supported by several morphological characters e.g., multiporous pore plates, large bilateral lophophores with high tentacle numbers (more than 30) and a vestibular collar (Schwaha, Winston, et al., 2022).A close relationship of S. sibogae to the multiporate F. hispida was recently indicated by a phylogenetic analysis based on the nuclear marker 18S gene (Schwaha, Waeschenbach, et al., 2022).Remarkably, S. sibogae is confirmed in Singapore well as in Brazil by our study and thereby underlines its vast distribution.S. sibogae was reported from Indonesia, Singapore, the eastern and western coast of Africa and the Western Atlantic before (Marcus, 1937(Marcus, , 1941;;Harmer, 1915;Schwaha, Winston, et al., 2022;Vieira et al., 2014).The only multiporate genus not included in our study is Elzerina, which is currently placed in the family Flustrellidridae.However, the presence of pseudocyphonautes larvae in Elzerina (like in F. hispida) is not confirmed but internal brooding of lecithotrophic larvae seems possible.Since an intertentacular organ is only present in Elzerina and neither in Flustrellidra nor in Pherusella, the latter two genera may share a closer relationship (Schwaha, 2021).Consequently, future studies should include sequence data of the genus Elzerina to confirm this idea.
Our study also suggests a sister-group relationship between the genus Alcyonidium and M. ambulans.The latter is a solitary bryozoan species living in sandy marine sediments as part of the meiofauna and was just recently rediscovered (Remane, 1936;Schwaha et al., 2024).Monobryozoidae was traditionally placed either among arachnidioids, primarily due to the presence of non-kenozooidal cystid appendages (Jebram, 1986), or as incertae sedis (D'Hondt, 1983).
More recent investigations suggest an affinity of monobryozoids with alcyonidioids (Schwaha, 2020c;Schwaha et al., 2024), which is also confirmed in our study.This affinity is reflected by alcyonidioidlike characters such as a circular orifice, the presence of a prominent orifical sphincter and a vestibular anus (Schwaha, 2020c;Schwaha et al., 2024).
The third main clade (C) includes species of four different ctenostome superfamilies, two of them are characterized by kenozooidal stolons as found in penetrantiids.The origin of cheilostomes within ctenostomes is the most accepted scenario and supported by morphological and molecular data and thereby renders ctenostomes paraphyletic (Orr et al., 2022;Waeschenbach et al., 2012).
However, it was still unclear which of the Recent ctenostome clades is the closest relative to cheilostomes.Former investigations suggested a close relationship and potential ancestry of cheilostomes with Arachnidium-like ctenostomes (Banta, 1975;Taylor, 1986Taylor, , 1990)).More recent studies favor a sister-group relationship of cheilostomes to the ctenostome superfamilies Hislopioidea and Vesicularioidea (Waeschenbach et al., 2012).Our study suggests a similar sister-group relationship of cheilostomes; however, additionally includes representatives of Walkerioidea and Victorellidae, which were not included in Waeschenbach et al. (2012).Thus, it seems reasonable that cheilostomes and the superfamilies in clade C (Walkerioidea, Victorellidae, Hislopioidea, Vesicularioidea) share a most recent common ancestor.Future studies should continue to tackle this question by increasing taxon sampling especially including more representatives of walkerioid and hislopioid bryozoans.
Regarding the sister-group relationship of Victorelloidea and Vesicularioidea, it is evident that they share a well-developed funicular system and a cardiac constrictor often with a gizzard (Schwaha, 2020c).However, while vesicularioid bryozoans are characterized by zooids that always are connected by true kenozooidal stolons, victorelloids lack stolons and are restricted to brackish and freshwater habitats (excluding sundanellids) (Schwaha, 2020c).
Based on morphological characters, the superfamily Victorelloidea was previously considered to be polyphyletic, which is supported in our analysis by the placement of Sundanella within Multiporata (see also Schwaha, Waeschenbach, et al., 2022;Schwaha, Winston, et al., 2022).Consequently, a morphological revision of Victorelloidea, with the exclusion of Sundanella, may reveal additional shared characters.In our analysis, A. epiphylla clusters together with the remaining two victorelloid species, with B. abscondita as sister taxon and T. muelleri being the sister taxon to both aforementioned.Formerly, A. epiphylla was regarded as cheilostome bryozoan due to the presence of an opercular-like structure (Metcalfe et al., 2007).Recent morphological investigations proved typical ctenostome features (denticulate gizzard, low tentacle numbers (eight), a large number of interzooidal pore plate cells and the lack of duplicature bands) and indicate a potential affinity with vesicularioids and victorelloids (Schwaha, Waeschenbach, et al., 2022).An operculum was not confirmed in A. epiphylla and therefore assumed to be absent (Schwaha, Waeschenbach, et al., 2022).Furthermore, a phylogenetic analysis based on the 18S gene revealed its ctenostome affinity (Schwaha, Waeschenbach, et al., 2022), which is also confirmed in our analysis.Since this species was only reported from mangroves it may be adapted to brackish environments with changeable salinities, which again might cohere with a victorelloid affiliation of A. epiphylla (Metcalfe et al., 2007;Schwaha, 2020c;Schwaha, Waeschenbach, et al., 2022).
Within Vesicularioidea, A. gracilis is the sister taxon to all remaining Amathia species as well as to V. spinosa.Amathia gracilis was previously placed in the genus Bowerbankia and just recently reassigned to Amathia (Waeschenbach et al., 2015).

| A ctenostome affiliation of Penetrantiidae and their closest relatives
Our analysis confirms a ctenostome affiliation of Penetrantiidae as suggested by several morphological studies previously (Decker et al., 2023;Pohowsky, 1978;Schwaha, 2020c;Silén, 1946Silén, , 1947)), and contradicts other studies that favored a cheilostome affinity (Smyth, 1988;Soule & Soule, 1969).Especially, the presence of cheilostome-like features such as the operculum and the brood chamber started a long-lasting discussion on the placement of penetrantiids.However, these structures appear to have evolved convergently in Penetrantiidae and Cheilostomata, since there are major morphological differences, particularly in the underlying musculature (see Decker et al., 2023).
Additionally, the absence of opercula and brood chambers in the closely related taxa (Paludicella, Terebripora and Arachnidium) points to apomorphic characters of Penetrantiidae.Paludicella pentagonalis differs in its colony pattern from P. articulata, in contrast to the P. articulata, P. pentagonalis has a linear series of zooids with no lateral branches (Annandale, 1916).Paludicella pentagonalis is also reported to sometimes possess "stolon-like" connections between zooids (see Rogick & Brown, 1942) that might support a potential relationship of P. pentagonalis with arachnidioids or penetrantiids.Therefore, it would be essential to investigate whether these stolon-like tubes feature pore plates because no information is currently available about the kenozooidal status of these tubes limiting their phylogenetic value.
The presence of true polymorphic stolons in penetrantiids traditionally favored a close relationship with the other stolonbearing groups vesicularioids or walkerioids (Decker et al., 2023;Schwaha, 2020c).The potential presence of a gizzard also supported a vesicularioid affinity (Pohowsky, 1978;Schwaha, 2020c;Silén, 1946Silén, , 1947)).However, the other two stolonate groups are not considered closely related to penetrantiids and also do not form a monophyletic group.How terebriporids fit into this clade remains questionable, but they maybe lack true stolons after all, which could explain the unexpected close relationship with Arachnidium (see above).Consequently, our study suggests that kenozooidal stolons have evolved at least three times independently within ctenostomes (vesicularioids, walkerioids and penetrantiids).The polyphyly of the artificial construct of "Stolonifera" was already suggested (Jebram, 1973;Schwaha, 2020c) and is also supported by recent molecular studies (Waeschenbach et al., 2012(Waeschenbach et al., , 2015)).This hypothesis is also based on several morphological and ontogenetical differences in the stolons of these two taxa (see Jebram, 1973;Schwaha, 2020c).
Stolon-like structures are also present in other groups of bryozoans and essential in the formation of characteristic colony forms and in the interaction and competition between other sessile organisms (Pohowsky, 1978;Schack et al., 2019).Stolons might also support faster propagation and expansion of colonies, ensuring good colony interconnectivity and the distribution of metabolites throughout a colony (Jebram, 1973;Pohowsky, 1978).Growth experiments on Penetrantia showed that their stolons grow relatively fast, which probably helps them invade new substrates faster and outcompete other endolithic organisms (Decker et al., submitted).In endolithic bryozoans, the main advantage of stolonate colonies with spaced zooids probably lies in decelerated substrate deterioration, thereby ensuring substrate stability (Decker et al., 2023;Pohowsky, 1978).
This might explain why all endolithic bryozoans have relatively long kenozooidal stolons or long cystid appendages between their zooids.
Furthermore, the presence of a true gizzard in penetrantiids was questioned, as the gizzard-like structure is indistinct, does not feature denticles and thereby resembles a proventriculus (Decker et al., 2023).Overall, a closer relationship of penetrantiids and vesicularioids is unlikely.

| Interrelationship of Penetrantiidae
The sequences of nine different penetrantiid specimens correspond to eight genetically diverged species in our analysis.However, there are two cryptic species complexes present, which can be hardly differentiated based on morphological characters.Cryptic speciation is a common phenomenon known from many different groups of bryozoans becoming more evident with the increase of molecular investigations (Chimenz Gusso et al., 2004;Fehlauer-Ale et al., 2014;Thorpe & Ryland, 1979;Waeschenbach et al., 2015).Particularly, the soft bodied ctenostomes, without any distinct skeletal characters, are prone to this taxonomic issue (Thorpe et al., 1978;Waeschenbach et al., 2015).
We unraveled two cryptic species complexes within the genus Penetrantia: (1) a species complex in the North Sea and the Northern Atlantic and (2) parva complex in the Southern Pacific.The species assembly in the Northern Atlantic is intriguing since at least two similar species do co-occur in the same region (Roscoff, France), P. concharum and Penetrantia sp.Penetrantia concharum from Roscoff is genetically identical to P. concharum from Sweden while Penetrantia sp. from Roscoff is genetically very different from P. concharum and most likely represents an undescribed species.Although P. concharum and Penetrantia sp.do not form a monophyletic clade in our analysis, their morphology is very similar and they form almost identical borehole apertures and colonies.A recent study found minor soft body differences between Penetrantia sp. and P. concharum.For instance, Penetrantia sp.features a collar, has a thinner operculum and on average smaller autozooids than P. concharum from Sweden (see Decker et al., 2023).However, since these morphologically investigated specimens were not sequenced it is not possible to assign these characters to one species with certainty.Furthermore, there are reports of Penetrantia along the Iberian coast that were not assigned to one of the known European penetrantiid species (P.concharum or Penetrantia brevis) and might represent the undescribed species in Roscoff (Decker et al., 2023;Reverter-Gil et al., 1995, 2016;Reverter-Gil & Souto, 2014).The picture becomes even more complex as the undescribed Penetrantia sp. from Roscoff (France) is also confirmed in Helgoland (Germany), which is geographically much closer to Sweden than France, and suggests an overlapping distribution of both species in the North Sea and the Northern Atlantic.Consequently, a much more detailed analysis at population level is required to delineate Penetrantia species occurring in the Northern Atlantic that should also include specimens from Norway, United Kingdom, Belgium, Spain and Portugal.
The second cryptic species complex is the parva complex distributed throughout the Southern Pacific and represented by three specimens in our study (northern and southern Islands of New Zealand and Chile).Penetrantia parva was also reported from New Caledonia and Hawaii and has one of the largest distributions of penetrantiids (see table 2 in Decker et al., 2023).This species complex is morphologically characterized by unique borehole apertures with prominent apertural notches, heavy cuticularized opercula and gonozooids where the brood chamber is half as long as the gonozooid itself (Decker et al., 2023;Silén, 1946Silén, , 1947)).Interestingly, P. cf. parva from southern New Zealand is more closely related to the Chilean one than P. parva from northern New Zealand.As zooid dimensions are very similar, the only considerable difference is the presence of a shallow pit in the frontal side of the operculum in some specimens of P. parva from northern New Zealand.Since this pit was never observed in specimens of the remaining two representatives of the parva complex, it might indicate a more distant relationship between P. parva from northern New Zealand to both P. cf. parva from southern New Zealand and Chile (Decker et al., 2023).However, the genetic distances between specimens from all three localities are sufficient (>10%) to consider each of them as a separate species, when applying a cox1 genetic distance of more than 3% as the threshold for species delimitation (see Baptista et al., 2022).The threshold of genetic distance for species delimitation is, however, still debated and depends on the marker gene and the group of animals investigated, but a threshold of about 3% is considered to have the lowest error rate with an optimum of 2.6% for cowrie gastropods (Meyer & Paulay, 2005).

Similar cryptic speciation was observed in the cheilostome
Bugula neritina, which was considered to have a cosmopolitan dis- tribution, yet only one of the three cryptic species in this complex is distributed globally (Fehlauer-Ale et al., 2014).On an even smaller geographical scale, cryptic bryozoan species were discovered in a recent study focusing on Reteporella species from the Azores and Mediterranean Sea (Baptista et al., 2022).Accordingly, cryptic speciation in bryozoans seems to be unexplored with many cryptic species complexes awaiting their discovery.Considering that most bryozoans have short-living lecithotrophic larvae, including penetrantiids, the gene flow between populations might be rather restricted, and consequently, speciation may occur on smaller geographical scales (Decker et al., 2023;Gruhl, 2020;Reed, 1991;Todd et al., 1998).However, level of gene flow and genetic structure between populations are not solely explained by pelagic larvae duration (PLD), since there are many examples of species that have a restricted distribution despite having a long PLD and vice versa (Todd et al., 1998).
Similar to the cryptic species complex in the Northern Atlantic, future work should include more specimens from different locations and combine molecular results with thorough morphological investigations.Additionally, it might be important to apply different genetic markers and a larger dataset to better resolve cryptic speciation in Penetrantia and to better understand intra-and interspecific genetic diversity (see Baptista et al., 2022;Fehlauer-Ale et al., 2014).Despite large genetic distances, potential new cryptic penetrantiid species should be validated with mating trials to confirm whether they are truly different biological species or not (see Gomez et al., 2007).
Nevertheless, there are three penetrantiid species in our study (P.clionoides, P. irregularis and P. japonica sp.nov.) that do not form species complexes and are well separated from other penetrantiids on molecular and morphological basis.
Penetrantia clionoides from Guam is the sister taxon of the P. parva clade and differs in the morphology of its operculum and gonozooid from the latter.The operculum of P. clionoides has a rough crescentshaped area on its frontal side and is partially composed of calcium carbonate, which is otherwise only known from P. japonica sp.nov.
from Japan and P. bellardiellae from Papua New Guinea (Decker et al., 2023;Schwaha et al., 2019;Smyth, 1988).Although the latter three species (P.clionoides, P. bellardiellae and P. japonica sp.nov.) exhibit similar opercula, they clearly differ in terms of gonozooid shape and/or interval length between tubulets (Decker et al., 2023).The geographically closest species to Japan is Penetrantia taeanata from South Korea (Seo et al., 2018).This species is much smaller than

| Terebriporidae and the convergent evolution of the boring life style
In our analysis, the family Terebriporidae is represented by a sole species from Chile and is placed among arachnidioid ctenostomes.
The original type specimen of the family and genus, Terebripora ramosa, was also collected in Chile (d 'Orbigny, 1847;Pohowsky, 1978).
Characteristic tubulets arising from the zooids were reported for T. ramosa along with very symmetrical feeder-shaped colonies, such that our specimens closely resemble T. ramosa (Pohowsky, 1978).
However, as there is no information regarding soft body morphology of the latter species, we cannot assign these specimens to T. ramosa with certainty.In fact, there is a lot of confusion in the literature about the correct affiliation of many terebriporid species, particularly of fossils.The enantiomorphic apertures of boring traces of Immergentia and Terebripora can appear very alike and probably led to the wrong assignment of species (Pohowsky, 1978), e.g., Spathipora comma (Soule, 1950a) was previously assigned to Terebripora and there is still confusion whether Immergentia philippinensis Soule, 1950b is a terebriporid or immergentiid species (Bobin & Prenant, 1954;Pohowsky, 1978;Soule, 1950aSoule, , 1950b)).Accordingly, it is not easy to assign boring traces to a family without information on stolon and gut morphology.In general, an affiliation of boring traces to a family, genus or even a species should be treated carefully as these traces resemble the boring activity of an animal and not true morphological characters.Therefore, such assignments should be considered separate ichnotaxa instead of a true biological species (see Bertling et al., 2006;Decker et al., 2023;Rosso, 2008;Wisshak et al., 2019).The problem becomes even more apparent as the family Terebriporidae was erected based on boring traces and colony patterns alone without any soft body information, rendering the entire family an ichnotaxon (Bertling et al., 2006;d'Orbigny, 1847;Wisshak et al., 2019).Accordingly, a histological reinvestigation of the type material would be necessary to provide soft body information and confirm the taxonomic integrity of the family Terebriporidae.
How many times an endolithic life style has evolved independently remains unanswered.Such an adaptation most likely occurred convergently within the lineage leading to the family Penetrantiidae and within the Arachnidioidea, which includes the boring family Immergentiidae (Pohowsky, 1978;Schwaha, 2020c;Silén, 1947).The fourth Recent endolithic bryozoan family, Spathiporidae, is commonly assigned to vesicularioids and thereby not closely related to the other endolithic taxa, which indicates that the endolithic life style has evolved independently in this group.
Spathiporidae and Terebriporidae were considered closely related among the endolithic taxa, since they share unique tubulets arising from autozooids, which the other two families Penetrantiidae and Immergentiidae are lacking.The most distinct difference between spathiporids and terebriporids is the connection of the zooids to their stolonal network.While spathiporids have pedunculate zooids, terebriporids have their zooids placed along the stolons and lack a peduncle (Pohowsky, 1978;Schwaha, 2020c;Soule & Soule, 1975).
Considering that spathiporids are vesicularioids, an endolithic life style should have evolved at least two times independently in ctenostomes and probably an additional time within arachnidioids.
With the fossil record dating back to the Ordovician, an early radiation within different ctenostome lineages seems plausible (Pohowsky, 1978).
Since H. expansa use a specialized gnawing apparatus with teeth to mechanically bore into uncalcified tubes of polychaetes like Chaetopterus, its burrowing lifestyle probably evolved independently as well (Borg, 1940;Pröts et al., 2019;Schwaha, 2020c).Colonies of B. abscondita live inside degraded wood, which is yet another different substrate, and its burrowing lifestyle most likely evolved independently too, reflected in the separated placement and affiliation of B. abscondita to victorelloids (Braem, 1951;Schwaha, 2020c).
Overall, such lifestyles have evolved about five times independently within ctenostomes and probably even more often when taking all the different boring ctenostome taxa into account that are only known from the fossil records (Pohowsky, 1978).Such a lifestyle has also evolved multiple times convergently within other groups of invertebrates, such as mollusks and polychaetes.An endolithic lifestyle has evolved at least eight times independently within bivalves, manifested in a wide variety of shell morphologies, including chemical and mechanical borers (Collins et al., 2023).A similar pattern is observed in polychaetes with many boring representatives belonging to different families, which is also reflected in a wide variety of boring methods and constructions of their burrows (Çinar & Dagli, 2021).In sponges, endolithic forms are distributed among at least three different families, indicating a similar convergent radiation of boring species (Van Soest et al., 2012).The overall benefit of such a lifestyle is probably better protection against environmental stressors like waves and currents, but particularly to reduce predation pressure, which might constitute the main driver of the convergent evolution of an endolithic lifestyle in so many different taxa (Collins et al., 2023;Pohowsky, 1978).This holds particularly true for ctenostome bryozoans, which, in contrast to cyclostomes and cheilostomes, lack a calcified body wall (Schwaha, 2020b), thus providing additional protection to the delicate zooids when immersed into substrates (Pohowsky, 1978).However, a potential cost of such a lifestyle is the dependency on calcareous substrates, which limits the spatial and geographical distribution of such species (Collins et al., 2023).Many calcareous substrates that could potentially provide habitats for boring bryozoans, such as whale bones, have not been searched for boring bryozoans, which could still harbor a large diversity of boring bryozoans and other boring taxa.

| CON CLUS ION
This study provides the most comprehensive up-to-date phylogeny F I G U R E A 2 Bayesian Inference phylogenetic tree based on a data matrix of 16 genes comprising 28 ctenostomes, 9 cheilostomes from Orr et al., 2021 (branch collapsed) and the phylactolaemate bryozoan Pectinatella magnifica (from Fuchs et al., 2009;Gim et al., 2018;Waeschenbach et al., 2009) as an outgroup to root the phylogenetic tree (see Table A2).Values on internal nodes correspond to BI posterior probabilities.
TA B L E A 1 PCR primers used for PCR amplification.
cleotides.The best-fitting evolutionary model for each partition was estimated using ModelTest-NG v0.1.7(Darriba et al., 2019) based on the corrected Akaike Information Criterion.The GTR+I+G4 was the best-fitting model for the rRNA genes and the MtZoa+G4+F was the best-fitting model for the PCGs.The ML tree was inferred using RAxML-NG v. 1. 0. 2(Kozlov et al., 2019) using the best-fitting model for each partition as determined by ModelTest-NG.Topological support was assessed with 1000 bootstrapping replicates.The BI analysis was conducted with MrBayes5d 3.2.6 (https:// github.com/ astan abe/ mrbay es5d: last accessed on 26.02.2023), a modified version of MrBayes 3.1.2incorporating the MtZoa evolutionary model (Ronquist & Huelsenbeck, 2003).Analyses were composed of two independent runs with four Markov Chain Monte Carlo (MCMC) chains, each.Chains were run for five million generations.Tree and parameter sampling were every 100th generation.The GTR+I+G4 model was used to correct for multiple substitutions of the nuclear and mt rRNA gene partitions, and the MtZoa+G4 model was used for mt PCGs gene partitions.Convergence of the MCMC chains was assessed by inspection of the tracefile outputs in Tracer (Nascimento Figure A2).The phylogeny is robust, and most nodes are either fully supported (100 bootstrap (BS)/1.00Posterior Probability (PP)) or highly supported (>90 BS/>0.99PP); while only four nodes have moderate support (<80 BS).Hereafter, only supports below 100 BS and 1.00 PP will be mentioned as all remaining branches are fully supported.
Penetrantia japonica sp.nov. is the sister taxon to all other Penetrantia species in our study.The next branch is formed of Penetrantia irregularis from New Zealand and is well separated from the other New Zealand penetrantiids of the parva clade.With moderate support (80 BS/1.00 PP), Penetrantia sp. from France (Roscoff) is the sister taxon to a clade composed of Penetrantia concharum from Sweden and France (Roscoff), Penetrantia clionoides from Guam and representatives of the parva complex from Chile and New Zealand.Penetrantia clionoides is the sister taxon to the Penetrantia parva complex (Figure2).Both species from France possess concharumlike borehole apertures, which are typically kidney-shaped; however, F I G U R E 1 Maximum Likelihood phylogenetic tree based on a data matrix of 16 genes comprising 28 ctenostomes, 9 cheilostomes fromOrr et al., 2021 (branch collapsed)  and the phylactolaemate bryozoan Pectinatella magnifica (fromFuchs et al., 2009;Gim et al., 2018;Waeschenbach et al., 2009) as an outgroup to root the phylogenetic tree (see TableA2 ).Values on internal nodes correspond to ML bootstrap support (1000 replicates) and posterior probabilities for BI (based on the last 75% of trees) respectively.Values are only shown for nodes that are not fully supported by both phylogeny reconstruction methods.Different colored boxes represent different cladesorange: Gymnolaemata, blue: Penetrantia, green: Multiporata, pink: Victorellidae, yellow: Vesicularioidea.Gray: three main clades A, B and C. Clade B reflects the superfamily Alcyonidioidea.Cheilostomata has been collapsed to allow better visualization.The scale bar represents 1 substitutional change per 100 character positions.
study represents the broadest taxon sampling to date and resulted in three main clades of ctenostomes.The first main clade (A) includes representatives of four different families of ctenostome bryozoans (Paludicellidae, Arachnidiidae, Terebriporidae and Penetrantiidae).Although a close relationship between the superfamilies Arachnidioidea and Paludicelloidea was previously pro- of ctenostome bryozoans, including representatives of all commonly accepted ctenostome superfamilies.It corroborates the paraphyletic status of "Ctenostomata" by the inclusion of cheilostomes as the sister taxon of a clade comprising Walkerioidea, Victorellidae, Hislopioidea and Vesicularioidea.Furthermore, this study gives the first molecular support for a ctenostome affiliation of Penetrantiidae and reveals a potential sister-group relationship to a clade containing P. articulata, Arachnidium sp. and Terebripora sp.However, additional morphological investigations, particularly on P. pentagonalis are essential to better understand this close relationship.The same holds true for Terebripora, since its arachnidioid affiliation is still surprising, and morphological information about this family are urgently required, not only to better understand its phylogeny but also to solve the ichnotaxonomic issue.We also unraveled two cryptic species complexes, one in the North Sea and Northern Atlantic, and the parva complex in the Southern Pacific.Additionally, we confirm and describe P. japonica sp.nov.as a new species from Japan.Given the cryptic nature of these endolithic bryozoans, their potential diversity is expected to be much higher with many more boring species awaiting their discovery.Moreover, our study proposes a monophyletic nature of Alcyonidioidea, with the monophyletic Multiporata nesting firmly within the latter, including the family Sundanellidae.However, sequence data of the multiporate genus Elzerina, would be essential to completely confirm the monophyly of Multiporata.Since this study provides the first complete mt genomes of 27 different ctenostomes it contributes to recent and future studies on this cryptic group of bryozoans.Although, we cover most ctenostome superfamilies, some of them are underrepresented und future studies should include more species to better understand their interrelationships.This holds particularly true for: Arachnidioidea, Walkerioidea, Benedeniporoidea and the families Hislopiidae, Victorellidae and Alcyonidiidae.Once sequence data of all recent endolithic families is available, a future study should combine all data to infer how often this lifestyle evolved independently within ctenostomes.Circularized mitochondrial genome map of Penetrantia parva from northern New Zealand (a) and Penetrantia clionoides from Guam (b).Arrows show direction of transcription with outer strand corresponding to the forward and the inner to the reverse strand.